Reactivation of electric eel acetylcholinesterase (AChE) inhibited by MeO(NH2)P(O)SMe (methamidophos) and by MeS(NH2)P(O)SMe was studied at pH 7.5 and 25 degrees C. The former inhibited enzyme shows a rather rapid spontaneous reactivation (t1+2 = 3.7 h); this reactivation is accelerated by 1 micrometer of the bispyridinium oximes TMB4 and obidoxine, and, to a lesser extent, by the monopyridinium oximes P2S and its 1-benzyl analogue (benzyl-P2A). The latter inhibited enzyme shows rapid aging (t1+2 = 0.6 h). Reactivation with 1 mM of the bispyridinium oximes is incomplete and reactivation with 1 mM of the monopyridinium oximes proceeds very slowly. These large differences between the properties of the two inhibited enzymes indicate that the methylthio group is the leaving group during inhibition of AChE by methamidophos. Additional support is afforded by the observation of induced aging of the former inhibited enzyme by thiourea. Upon comparison of the reactivation of AChE inhibited by methamidophos with that of AChE inhibited by an N-methyl analogue, crufomate, and an N,N-dimethyl analogue, tabun, it appears that the rate of spontaneous reactivation decreases with increasing alkylation of the P-NH2 group. Whereas benzyl-P2A is somewhat less active than P2S for reactivation of ACHE inhibited by methamidophos, it is superior to P2S for reactivation of ACHE inhibited by crufomate and also superior to P2S and to the bispyridinium oximes for AChE inhibited by tabun.